Bottom Line:
After extraction of the kettin-associated actin, the A-band edges were also stained.Dotblot analysis revealed binding of COOH-terminal kettin domains to myosin.We conclude that kettin is attached not only to actin but also to the end of the thick filament.

Affiliation: Institute of Physiology and Pathophysiology, University of Heidelberg, D-69120 Heidelberg, Germany.

ABSTRACTKettin is a high molecular mass protein of insect muscle that in the sarcomeres binds to actin and alpha-actinin. To investigate kettin's functional role, we combined immunolabeling experiments with mechanical and biochemical studies on indirect flight muscle (IFM) myofibrils of Drosophila melanogaster. Micrographs of stretched IFM sarcomeres labeled with kettin antibodies revealed staining of the Z-disc periphery. After extraction of the kettin-associated actin, the A-band edges were also stained. In contrast, the staining pattern of projectin, another IFM-I-band protein, was not altered by actin removal. Force measurements were performed on single IFM myofibrils to establish the passive length-tension relationship and record passive stiffness. Stiffness decreased within seconds during gelsolin incubation and to a similar degree upon kettin digestion with mu-calpain. Immunoblotting demonstrated the presence of kettin isoforms in normal Drosophila IFM myofibrils and in myofibrils from an actin- mutant. Dotblot analysis revealed binding of COOH-terminal kettin domains to myosin. We conclude that kettin is attached not only to actin but also to the end of the thick filament. Kettin along with projectin may constitute the elastic filament system of insect IFM and determine the muscle's high stiffness necessary for stretch activation. Possibly, the two proteins modulate myofibrillar stiffness by expressing different size isoforms.

Mentions:
To characterize the sarcomere length (SL)-tension relationship of nonactivated Drosophila IFM, force measurements were performed on single myofibrils (Fig. 1) . The force of three to four myofibrils of similar length was recorded in identical step-stretch protocols, and median-filtered force traces were superimposed to obtain clearer signals (Fig. 1 B). A summary of stress-strain curves obtained from 24 myofibrils is shown in Fig. 1 C. Because stretched myofibrils showed inhomogeneous SLs, Fig. 1 C depicts passive tension related either to the length of the longest sarcomere (solid line) or to mean SL (dotted line). At larger stretches, the difference between the two curves was significant. Passive tension rose steeply on low stretch and reached a first plateau after ∼5% extension. Additional stretch further increased force until at ∼12% extension a second plateau phase (or some force decline) commenced. Higher stretches frequently led to myofibril breakage. Upon release of myofibrils from the stretched SL, large force hysteresis was seen.

Mentions:
To characterize the sarcomere length (SL)-tension relationship of nonactivated Drosophila IFM, force measurements were performed on single myofibrils (Fig. 1) . The force of three to four myofibrils of similar length was recorded in identical step-stretch protocols, and median-filtered force traces were superimposed to obtain clearer signals (Fig. 1 B). A summary of stress-strain curves obtained from 24 myofibrils is shown in Fig. 1 C. Because stretched myofibrils showed inhomogeneous SLs, Fig. 1 C depicts passive tension related either to the length of the longest sarcomere (solid line) or to mean SL (dotted line). At larger stretches, the difference between the two curves was significant. Passive tension rose steeply on low stretch and reached a first plateau after ∼5% extension. Additional stretch further increased force until at ∼12% extension a second plateau phase (or some force decline) commenced. Higher stretches frequently led to myofibril breakage. Upon release of myofibrils from the stretched SL, large force hysteresis was seen.

Bottom Line:
After extraction of the kettin-associated actin, the A-band edges were also stained.Dotblot analysis revealed binding of COOH-terminal kettin domains to myosin.We conclude that kettin is attached not only to actin but also to the end of the thick filament.

Affiliation:
Institute of Physiology and Pathophysiology, University of Heidelberg, D-69120 Heidelberg, Germany.

ABSTRACTKettin is a high molecular mass protein of insect muscle that in the sarcomeres binds to actin and alpha-actinin. To investigate kettin's functional role, we combined immunolabeling experiments with mechanical and biochemical studies on indirect flight muscle (IFM) myofibrils of Drosophila melanogaster. Micrographs of stretched IFM sarcomeres labeled with kettin antibodies revealed staining of the Z-disc periphery. After extraction of the kettin-associated actin, the A-band edges were also stained. In contrast, the staining pattern of projectin, another IFM-I-band protein, was not altered by actin removal. Force measurements were performed on single IFM myofibrils to establish the passive length-tension relationship and record passive stiffness. Stiffness decreased within seconds during gelsolin incubation and to a similar degree upon kettin digestion with mu-calpain. Immunoblotting demonstrated the presence of kettin isoforms in normal Drosophila IFM myofibrils and in myofibrils from an actin- mutant. Dotblot analysis revealed binding of COOH-terminal kettin domains to myosin. We conclude that kettin is attached not only to actin but also to the end of the thick filament. Kettin along with projectin may constitute the elastic filament system of insect IFM and determine the muscle's high stiffness necessary for stretch activation. Possibly, the two proteins modulate myofibrillar stiffness by expressing different size isoforms.